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Chem. Res. Toxicol. 1990, 3, 144-149
DNA Damage Induced by Metabolites of o-Phenylphenol in the Presence of Copper(I1) Ion Sumiko Inoue, Koji Yamamoto, and Shosuke Kawanishi” Department of Public Health, Faculty of Medicine, Kyoto University, Kyoto 606, Japan Received July 10, 1989
Reactivities of o-phenylphenol and its metabolites (2,5-dihydroxybiphenyl, 2-phenyl-1,4benzoquinone) with DNA were investigated by a DNA sequencing technique, and the reaction mechanism was studied by UV-visible and ESR spectroscopies. In the presence of Cu(II), 2,5-dihydroxybiphenyl caused strong DNA damage even without piperidine treatment. Catalase, methionine, and methional inhibited the DNA damage completely, whereas mannitol, sodium formate, ethanol, tert-butyl alcohol, and superoxide dismutase did not. 2,5-Dihydroxybiphenyl plus Cu(I1) frequently induced a piperidine-labile site a t thymine and guanine residues. The addition of Fe(III), Mn(II), Co(II), Ni(II), Zn(II), Cd(II), or Pb(I1) did not induce DNA damage with 2,5-dihydroxybiphenyl. When H202 was added, 2-phenyl-l,4-benzoquinone also induced DNA damage in the presence of Cu(I1). Cu(I1) accelerated the autoxidation of 2,5-dihydroxybiphenyl to quinone. An ESR study revealed that the semiquinone radical is an intermediate of the autoxidation. Catalase had no inhibitory effect on the acceleration by Cu(I1). Superoxide dismutase promoted both the autoxidation of 2,5-dihydroxybiphenyl and the initial rate of semiquinone radical production. ESR spin trapping experiments showed that the addition of Fe(II1) produced hydroxyl radical during the autoxidation of 2,5-dihydroxybiphenyl, whereas the addition of Cu(I1) hardly did so. The results suggest that DNA damage by 2,5-dihydroxybiphenyl plus Cu(I1) is due to active species other than hydroxyl free radical.
Introduction OPP’ and its sodium salt have been used as fungicides for citrus fruits (1). It has been reported that long-term administration of OPP and its sodium salt at high dose induces carcinoma of the urinary bladder in rats (1-6). There is a report that the sodium salt of OPP showed a promoting effect on urinary bladder carcinogenesis initiated by a nitrosamine derivative (7). OPP and its sodium salt have mt been proved to be mutagenic in bacterial test systems (1,8),although Hiraga et al. reported that OPP has genotoxicity in CHO-K1 cells and in a human cell strain (9,lO). Reitz et al. proposed that the bladder tumors are induced by a nongenetic mechanism (8). It is widely accepted that many carcinogens are readily converted to reactive intermediates by drug-metabolizing enzymes. The metabolites of OPP in the free form include Di-OH-BP and PBQ (8,11,12). Morimoto et al. reported that DNA damage was induced in urinary bladder epithelium of male rats treated with PBQ (13). However, the mechanisms of DNA damage remain to be clarified. We examined the induction of DNA damage by metabolites of OPP using 32P-5’-end-labeledDNA fragments of defined sequences obtained from protooncogene (cHa-rus-1) and found that, in the presence of Cu(II), DiOH-BP and PBQ plus HzOzcaused strong DNA damage. Experimental Procedures Materials. Restriction enzymes (BstEII, AuaI, XbaI, PstI) and T4polynucleotide kinase were purchased from New England Biolabs. Calf intestine phosphatase was from Boehringer Manheim GmbH. [@%’]ATP (6000 Ci/mmol) was from New England Nuclear. CuCl, and other metallic chlorides, OPP, ethanol, tert-butyl alcohol, D-mannitol, and sodium formate were from Nakarai Chemicals Co., Kyoto, Japan. Di-OH-BP was from Tokyo * T o whom correspondence should be addressed.
Kasei Co., Tokyo, Japan. DTPA and bathocuproinedisulfonic acid were from Dojin Chemicals Co., Kumamoto, Japan. A solution of 30% H202was from Santoku Chemical Industries Co., Miyagi, Japan. Acrylamide, bis(acrylamide), and piperidine were from Wako Chemicals Co., Osaka, Japan. Hydrazine was from Eastman Organic Chemicals. SOD (3000 units/mg from bovine erythrocytes), catalase (45000 units/mg from bovine liver), and methional were from Sigma Chemical Co. PBQ, DMPO, and dimethyl sulfate were purchased from Aldrich Chemical Co. Ethanol solutions of OPP, Di-OH-BP, and PBQ were made up fresh each time. Preparation of 3’T-5’-End-LabeledDNA Fragments. DNA fragments were prepared from plasmid pbcNI which carries a 6.6-kilobase BamHI chromosomal DNA segment containing human c-Ha-ras-1 protooncogene (14). A 602-base-pair AuaI fragment (AuaI 1645-AuaI 2246) and a 435-base-pair AuaI fragment (Am1 2247-AuaI 2681) were obtained by subcloning and labeled at the 5’-termini with [ Y - ~ ~ P ] Aand T P T, polynucleotide kinase as previously described (14). The 3P-5’-end-labeled 602-base-pair AuaI fragment was again digested with XbaI to obtain a singly labeled 261-base-pair fragment (AuaI*1645-XbaI 1905) and a singly labeled 341-base-pair fragment (XbaI 1906-AuaI*2246), The 32P-5’-end-labeled435-base-pair AuaI fragment was digested with PstI to obtain a singly labeled 98-base-pair fragment (AuaI*2247-PstI 2344) and a singly labeled 337-base-pair fragment (PstI 2345-AuaI*2681). The asterisk indicates that 32Plabeling and nucleotide numbering starts with the BamHI site (15). Detection of DNA Damage Induced by OPP Metabolites. The standard reaction mixture in a microtube (1.5-mL Eppendorf) contained 0.1 mM OPP or its metabolites, 10 pM CuC12,and the [32P]DNAfragment (-0.5 fiM DNA nucleotide concentration) in 200 pL of 10 mM sodium phosphate buffer (pH 7.9) containing 5 WMDTPA. Since buffers and reagents are known to be invariably contaminated with trace amounts of metal ions (16), Abbreviations: OPP, o-phenylphenol; Di-OH-BP,2,5-dihydroxybiphenyl; PBQ, 2-phenyl-1,4-benzoquinone; DTPA, diethylenetriaminepentaacetic acid; SOD, superoxide dismutase; ESR, electron spin resonance; DMPO, 5,5-dimethylpyrrolineN-oxide; DMPO-OH, hydroxyl radical adduct of 5,5-dimethylpyrroline N-oxide.
0893-228x/90/2703-0144$02.50/0 0 1990 American Chemical Societv
DNA Damage by o-Phenylphenol Metabolites
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Figure 1. Autoradiogram of 32P-labeledDNA fragments incubated with OPP metabolites. Reaction mixture contained 32Plabeled 261-base-pair DNA fragment (AuaI*1645-XbaI*1905) and 0.1 mM OPP or its metabolites in 200 pL of 10 mM sodium phosphate buffer a t pH 7.9 containing 5 pM DTPA. Lane 1, control; lane 2, OPP; lane 3, PBQ; lane 4, Di-OH-BP; lane 5,lO pM CuC1,; lane 6, OPP + Cu(I1); lane 7, PBQ + Cu(I1); lane 8, Di-OH-BP + Cu(I1); lane 9, Di-OH-BP + Cu(I1) + 5 pM bathocuproine; lane 10, Di-OH-BP + Cu(I1) + 10 pM bathocuproine; lane 11, Di-OH-BP + Cu(I1) + 20 pM bathocuproine. After incubation for 10 min a t 37 "C, cold ethanol was added. The precipitated DNA fragments were electrophoresed on an 890 polyacrylamide/8 M urea gel, and the autoradiogram was obtained by exposing X-ray film to the gel. DTPA was added. The mixtures were incubated for 10 min a t 37 "C,followed by heating a t 90 "C for 20 min in 1M piperidine where indicated. The DNA fragments were electrophoresed on a 12 cm X 16 cm slab gel, and the autoradiograms were obtained by overnight exposure of X-ray film to the gels a t -85 "C as previously described (14, 17, 18). The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides with those produced by the chemical reactions of the Maxam-Gilbert procedure (19) by using a DNA sequencing system (LKB2010 Macrophor). A laser densitometer (LKB 2222 UltroScan XL) was used for the measurement of the relative amounts of oligonucleotides from treated DNA fragments. The intensity of absorbance was dependent on the exposure time of film and the specific activity of the [32P]DNAfragment used. UV-Visible Spectra Measurements. UV-visible spectra were measured a t 37 "C with a UV-vis near-IR recording spectrophotometer (Shimadzu UV-365). ESR Spectra Measurements. ESR spectra were measured a t room temperature by using a JES-FE-3XG (JEOL, Tokyo, Japan) spectrometer with 100-kHz field modulation according to the method previously described (20). Spectra were recorded with a microwave power of 16 mW and a modulation amplitude of 1.0 G. The magnetic fields were calculated by the splitting of Mn2+in MgO (AHM = 86.9 G). DMPO was used as a radical trapping reagent, when necessary. CuC12,FeC1, catalase, and/or SOD was added where indicated.
Results Damage of --Labeled DNA Fragments Induced by OPP Metabolites. The extent of DNA damage was estimated by gel electrophoretic analysis. Figure 1 shows an autoradiogram of DNA fragments treated with OPP and its metabolites in the presence or absence of Cu(I1). In the case of Di-OH-BP plus Cu(II), oligonucleotides were clearly detected on the autoradiogram as a result of DNA cleavage. The two bands in the control show doublestranded and single-stranded forms of DNA fragment. DNA cleavage increased with time (Figure 2A,B) and with
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 145
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Figure 2. Effects of piperidine treatment, incubation time, and concentration of Di-OH-BP on DNA cleavage in the presence of Cu(I1). The reaction mixture contained the 32P-5'-end-labeled 261-base-pair fragment (AuaI*1645-XbaI 1905),0.1 mM (A and B) or the indicated concentrations (C) of Di-OH-BP, and 10 pM CuC12 in 200 pL of 10 mM phosphate buffer a t pH 7.9 containing 5 pM DTPA. After the incubation for the indicated duration (A and B) or 10 min (C) a t 37 "C, followed by the piperidine treatment (A) or without the piperidine treatment (B and C), the DNA fragments were electrophoresed on an 890 polyacrylamide/8 M urea gel and the autoradiogram was obtained by exposing X-ray film to the gel. (A) Lane 1,O min; lane 2,lO min; lane 3,20 min; lane 4,30 min. (B) Lane 1 , O min; lane 2,lO min; lane 3,20 min; lane 4,30 min. (C) Lane 1,O mM Di-OH-BP; lane 2,0.05 mM Di-OH-BP; lane 3,O.l mM Di-OH-BP; lane 4,0.25 mM Di-OH-BP; lane 5,0.5 mM Di-OH-BP.
increasing concentration of Di-OH-BP (Figure 2C). The cleavage without piperidine treatment suggests the breakage of the deoxyribose-phosphate backbone by DiOH-BP plus Cu(I1). The amount of oligonucleotides increased with piperidine treatment (Figure 2A), suggesting that the base alteration(s) and/or liberation(s) were induced by Di-OH-BP plus Cu(I1) and subsequently the cleavages at those bases occurred. No cleavage was observed without Cu(II), nor with OPP and PBQ even in the presence of Cu(I1). Cu(I1) alone caused no DNA damage. The effect of bathocuproine, a Cu(1)-specific chelating agent, is shown in Figure 1. The addition of bathocuproine equal to or more than Cu(I1) completely inhibited DNA damage by Di-OH-BP plus Cu(I1) (lanes 10 and 11). The effects of other metal ions on Di-OH-BP-dependent DNA damage were examined (data not shown). Mn(II), Fe(III), Co(II), Ni(II), Zn(II), Cd(II), and Pb(I1) did not induce DNA damage in the presence of Di-OH-BP. Effects of Scavengers on Di-OH-BP plus Cu(I1)Induced DNA Damage. Figure 3 shows the effects of radical scavengers, SOD, and catalase on Di-OH-BP plus Cu(I1) induced DNA damage. Methional and methionine completely inhibited DNA damage (lanes 2 and 3), whereas hydroxyl radical scavengers (sodium formate, mannitol, ethanol, tert-butyl alcohol) and SOD did not. Catalase inhibited DNA damage completely (lane 9), suggesting the involvement of H202. Boiled catalase did not show the inhibitory effect (lane 10). The addition of H202enhanced DNA damage (data not shown). SOD did not induce DNA damage with Di-OH-BP in the absence of Cu(I1). Effects of H202 on OPP Metabolite Dependent DNA Damage. Since Di-OH-BP is supposed to be autoxidized to generate H202,the extent of DNA damage by Di-OH-BP plus Cu(I1) was compared with DNA damage by H202plus Cu(I1). Di-OH-BP plus Cu(I1) caused DNA
Inoue et al.
146 Chem. Res. Toxicol., Vol. 3,No. 2, 1990
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Figure 3. Effects of scavengers on DNA dama e by Di-OH-BP plus Cu(I1). The reaction mixture contained the &-5’-end-labeled 261-base-pairfragment (AuaI*1645.-XbaI 1905), 0.1 mM Di-OHBP, and 10 pM CuC1, in 200 pL of 10 mM phosphate buffer a t pH 7.9 containing 5 pM DTPA. Scavenger was added where indicated. Lane 1, no scavenger; lane 2,O.l M methional; lane 3,O.l M methionine; lane 4,O.l M mannitol; lane 5,O.l M sodium formate; lane 6, 2% ethanol; lane 7,270 tert-butyl alcohol; lane 8,30 units of SOD; lane 9,30 units of catalase; lane 10,30 units of boiled catalase. After incubation for 10 min a t 37 OC, the DNA fragments were analyzed by the method described in the Figure 1 legend.
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B
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Figure 5. Autoradiograms of DNA cleavages b Di-OH-BP plus Cu(I1) and PBQ plus H202plus Cu(II). (A) The h-8-end-labeled 98-base-pair fragment (AuaI*2247-PstI 2344) in 200 p L of 10 mM sodium phosphate buffer a t pH 7.9 containing 5 p M DTPA was incubated with 50 pM Di-OH-BP plus 10 pM CuCl, for 5 min a t 37 “C (lane 1). (B) The 3?P-5’-end-labeled 341-base-pair fragment (XbaI 1906-AuaI*2246) in 200 pL of 10 mM sodium phosphate buffer a t pH 7.9 containing 5 pM DTPA was incubated with 50 pM PBQ plus 50 pM H202plus 10 pM CuC12 (lane 1)or with 50 pM Di-OH-BP plus 10 pM CuC12(lane 2) for 5 min a t 37 “C. After the piperidine treatment, the DNA fragment was electrophoresed on an 8% polyacrylamide/8 M urea gel and the autoradiogram was obtained by exposing film to the gel. The lanes G represent the patterns obtained for the same fragment after cleavage a t guanine by the chemical methods of Maxam and Gilbert (19). Figure 4. Effect of H202 on O P P metabolite-dependent DNA damage. The reaction mixture contained the 32P-5’-endlabeled 337-base-pair fragment (PstI 2345-AuaI*2681), 0.1 mM OPP or its metabolite, and 10 pM CuC12 in 200 pL of 10 mM phosphate buffer a t pH 7.9 containing 5 pM DTPA. Where indicated, 0.1 mM H202 was added. Lane 1, CuC1, + H202;lane 2, Di-OH-BP + CuCl,; lane 3, PBQ + CuC1, + H202;lane 4, PBQ + H202;lane 5, OPP + H202+ CuC12. After incubation for 10 min a t 37 “C, the treated DNA fragments were analyzed by the method described in the Figure l legend.
and guanine residues in sequences containing many cytosine residues. Although the extent of cleavage at the cytosine positions was variable according to the sequence, central cytosine residues of the 5’-CCA-3’ sequence seemed to be cleaved extensively. The cleavages at or near to 12th codon of c-Ha-ras-1 protooncogene were not so strong under the present conditions (Figure 6B). Site specificity plus Cu(I1) of DNA damage induced by PBQ plus H202 was similar to the case of Di-OH-BP plus Cu(I1) (Figure
damage more efficiently than H202 plus Cu(I1). In the presence of H202,PBQ plus Cu(I1) caused DNA damage efficiently (Figure 4, lane 3). Site Specificity of DNA Damage Induced by DiOH-BP plus Cu(I1). For measurement of the relative intensity of DNA cleavages by Di-OH-BP plus Cu(II), 32P-5’-end-labeledDNA fragments incubated with DiOH-BP plus Cu(II), followed by the piperidine treatment, were electrophoresed and an autoradiogram was obtained as shown in Figure 5. Autoradiograms were scanned with a laser densitometer (Figure 6). The DNA cleavage sites were determined by using the Maxam-Gilbert procedure (18). Di-OH-BP plus Cu(I1) frequently induced a piperidine-labile site at thymine and guanine residues, especially those located between purine residues, but not at thymine
UV-Visible Spectroscopic Studies on the Autoxidation of Di-OH-BP. Figure 7 shows changes in the UV-visible spectra of Di-OH-BP plus Cu(I1) with time. When a stock solution of Di-OH-BP was added to a buffer solution, the solution turned yellow gradually with an increase of the absorption maximum at 366 nm. The absorption maximum at 366 nm can be attributed to PBQ. The addition of Cu(I1) accelerated the autoxidation of Di-OH-BP. Catalase did not inhibit the accelerating effect of Cu(I1). SOD promoted the autoxidation of Di-OH-BP, and boiled SOD showed no effect (data not shown). The overnight incubation of PBQ in a buffer solution caused disappearance of the absorption maximum at 366 nm, indicating that PBQ was gradually converted into other products.
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D N A Damage by o-Phenylphenol Metabolites
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